Bispecific antibody fragments for neurological disease proteins and methods of use

ABSTRACT

The present invention relates to therapeutic agents comprising bispecific recombinant antibody fragments to selectively clear a protein associated with a neurological disease and methods of use of these therapeutic agents to treat neurological diseases.

RELATED APPLICATIONS

This application is a 371 National Stage Application of InternationalApplication Number PCT/US2013/021032, filed Jan. 10, 2013, which claimspriority to U.S. Provisional Application No. 61/585,539 that was filedon Jan. 11, 2012, the contents of which are incorporated by referenceherein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 30, 2015, isnamed 17555.006US1_SL.txt and is 8,353 bytes in size.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is characterized by the presence of numerousneuritic plaques, neurofibrillary tangles, and neuronal loss. Theplaques, mainly composed of β-amyloid (Aβ) peptide fragments, arederived from the processing of amyloid precursor protein (APP) by β- andγ-secretases. The presence of reactive microglia, astrocytes, andcomplement factors associated with the fibrillar Aβ plaques suggests thedevelopment of a local and chronic inflammatory response within theplaque area and is consistent with the hypothesis that complementactivation contributes to this inflammatory process. In Aβ brains, C1q,the first component of the classical complement pathway that bindsfibrillar Aβ and activates complement, has been shown to be associatedwith fibrillar Aβ plaques. Other complement proteins have also beendetected in the plaque area, and their synthesis has been shown to occurwithin the AD brain.

Aggregation and deposition of amyloid β-protein (Aβ or beta amyloid) isconsidered to be a primary pathological event in Alzheimer's disease(AD). The longer 42-43 amino acid Aβ forms have been implicated in theformation of amyloid plaques, the aggregation state of the peptide iscritical in determining its neurotoxicity. Many different forms of Aβhave been identified and characterized including fibrils, proto-fibrils,annular structures, globular structures, amorphous aggregates andvarious soluble oligomers. Numerous studies indicate that smalloligomeric morphologies of Aβ are the primary toxic species in AD. Thesesmall oligomers are also called “low-n oligomers” (i.e., dimers,trimers, or tetramers).

Accordingly, there exists the need for new therapies and reagents forthe treatment of Alzheimer's disease, in particular, therapies andreagents capable of effecting a therapeutic and diagnostic benefit atphysiologic (e.g., non-toxic) doses.

SUMMARY OF THE INVENTION

The present invention relates to therapeutic agents comprisingbispecific recombinant antibody fragments to selectively clear proteinsassociated with neurological diseases, and methods of use of thesetherapeutic agents to treat neurological disease.

Certain embodiments of the present invention provide a bispecificantibody fragment comprising (a) a first ligand that specificallyrecognizes a protein associated with a neurodegenerative disease, and(b) a second ligand that specifically activates a classical complementpathway component or directly activate microglial cells.

Certain embodiments of the present invention provide a bispecificantibody comprising (a) a first scFv that is an H1 scFv specific forβ-amyloid, wherein the first scFv has an amino-terminus and acarboxy-terminus, and (b) a second scFv, that is an scFv specific forC1q, wherein the second scFv has an amino-terminus and acarboxy-terminus, and (c) a (Gly₄Ser)₃ peptide linker (SEQ ID NO: 1)operably linking the carboxy-terminus of the first scFv to theamino-terminus of the second scFv.

Certain embodiments of the present invention provide a nucleic acidencoding a bispecific antibody described above.

Certain embodiments of the present invention provide a therapeuticcomposition comprising a bispecific antibody described above, incombination with a physiologically-acceptable, non-toxic vehicle.

Certain embodiments of the present invention provide a method ofclearing aggregated and soluble Aβ comprising administering thebispecific antibody described above.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B. Increased Aβ levels in APP mice with age. Brains fromAPP transgenic mice and wild type control mice at 6, 12 and 24 monthsold were homogenized and dissolved by formic acid. Aβ40 (panel A) andAβ42 (panel 2B) concentrations were measured by Sandwich ELISA.

FIG. 2. Schematic diagram showing diabody s gene construct(pCantab6/Aβ+C1q). pelB, signal peptide sequence of bacterialpectatelyase; c-myc, a sequence encoding an epitope recognized by themonoclonal antibody 9E10; His6, a sequence encoding six C-terminalhistidine residues (SEQ ID NO: 2).

FIG. 3. BIAcore binding analysis of diabody showing binding to Aβ andC1q. Phase I, diabody binding to immobilized Aβ chip. Phase II, C1qbinding to Aβ/diabody complex.

FIG. 4. Sorting of p6 peptide, left: no antigen; right: 100 nM antigen.Cells in quadrant 2 should bind target antigen. Boxed area showsselection criteria for sorted cells.

FIG. 5. Schematic of Atomic Force Microscope (AFM) biopanning protocol.

FIGS. 6A and 6B. The AFM image of formation of oligomeric (A) andfibrillar α-synuclein aggregates (B). The sample of monomeric or amixture of monomer and oligomeric α-synuclein were dissolved to a finalconcentration of 70 μM. To form fibrils, the sample was incubated at 56°C. for 12 days and then at 37° C. for 7 days without shaking and finallyincubated at 4° C. for 10 more days.

FIG. 7. Sequences for H1v2 anti-Ab40 (SEQ ID NO: 6), H1v3 anti-Ab40(also called D9antiAb40) (SEQ ID NO: 7), and C1 antiAb40 (SEQ ID NO: 8).The first two sequences are different variants that bind better than theoriginal H1 sequence. The C1antiAb40 scFv binds near the c-terminal ofAβ, C1. These sequences omit the PELB leader and the end NotI/His tags.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the present invention provides therapeuticagents comprising bispecific antibody fragments to selectively clearproteins associated with neurological diseases, and methods of use ofthese therapeutic agents to treat neurological diseases. Certainembodiments of the present invention provide a bispecific antibodyfragment comprising

(a) a first ligand that specifically recognizes a protein associatedwith a neurodegenerative disease, and

(b) a second ligand that specifically activates a classical complementpathway component or directly activate microglial cells.

In certain embodiments, the bispecific antibody fragment furthercomprises a second bispecific antibody fragment to form a diabodyconstruct. In certain embodiments, the first and second ligands are scFvfragments. In certain embodiments, the second ligand specifically bindsto C1q (i.e., the first component of the classical complement response).In certain embodiments, the second ligand specifically binds to areceptor on a leukocyte, such as CD16 or CD64.

In certain embodiments, the bispecific antibody fragment treats aneurodegenerative disease such as Alzheimer's disease, Parkinson'sdisease, Lou Gehrig's disease, Huntington's disease, a prion disease ora spongiform encephalopathy. In certain embodiments, theneurodegenerative disease Alzheimer's disease and the first ligand thatspecifically recognizes Aβ (e.g., the first ligand is specific forsoluble Aβ or is specific for aggregated Aβ). In certain embodiments,the bispecific antibody fragment further comprises a (Gly₄-Ser)₃ linker(SEQ ID NO: 1) operably linked between the first and second ligand. Incertain embodiments, the bispecific antibody fragment further comprisesa poly-His tail operably linked to either the first or second ligand.

Certain embodiments of the present invention provide a bispecificantibody comprising

(a) a first scFv that is an H1 scFv specific for β-amyloid, wherein thefirst scFv has an amino-terminus and a carboxy-terminus, and

(b) a second scFv that is an scFv specific for C1q, wherein the secondscFv has an amino-terminus and a carboxy-terminus

(c) a (Gly₄Ser)₃ peptide linker (SEQ ID NO: 1) operably linking thecarboxy-terminus of the first scFv to the amino-terminus of the secondscFv.

In certain embodiments, the bispecific antibody further comprises a gIIIsignal sequence operably linked to the amino-terminus of the first scFv.In certain embodiments, the bispecific antibody further comprises ac-myc tag operably linked to the carboxy-terminus of the second scFv. Incertain embodiments, the bispecific antibody further comprises a (His)₆tag (SEQ ID NO: 2) operably linked to the c-myc tag.

Certain embodiments of the present invention provide a nucleic acidencoding a bispecific antibody described above. In certain embodiments,the nucleic acid further comprises a promoter. Examples include, but arenot limited to, a lac promoter, the SV40 early promoter, mouse mammarytumor virus LTR promoter; adenovirus major late promoter (Ad MLP); aherpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, pol II promoters, pol III promoters, syntheticpromoters, hybrid promoters, and the like. In addition, sequencesderived from nonviral genes, such as the murine metallothionein gene,will also find use herein. Such promoter sequences are commerciallyavailable from, e.g., Stratagene (San Diego, Calif.).

In certain embodiments, other control elements, such as enhancers andthe like, will be of particular use. In certain embodiments, a gIIIsignal sequence is included at the 5′ terminus. In certain embodiments,the nucleic acid further comprises a nucleic acid encoding a c-myc tagand a nucleic acid encoding a (His)₆ tag (SEQ ID NO: 2) that arepositioned in-frame at the 3′ terminal of the bispecific antibody. ThegIII signal sequence directs the polypeptide into the periplasmic space,where it can fold correctly in a soluble form. The c-myc tag is used toanalyze the expression level of the bispecific scFv, and (His)₆ tag (SEQID NO: 2) can be used to purify the bispecific scFv protein.

Certain embodiments of the present invention provide an expressioncassette comprising the nucleic acid sequence described above and apromoter.

Certain embodiments of the present invention provide a vector comprisingthe expression cassette described above. In certain embodiments, thevector is a viral vector. In certain embodiments, the viral vector is anadenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV,or murine Maloney-based viral vector.

Certain embodiments of the present invention provide the vector orexpression cassette described above.

Certain embodiments of the present invention provide a therapeuticcomposition comprising a bispecific antibody described above, incombination with a physiologically-acceptable, non-toxic vehicle.

Certain embodiments of the present invention provide a method ofclearing aggregated and soluble Aβ comprising administering thebispecific antibody described above. In certain embodiments, theaggregated and soluble Aβ is in a cell, such as in brain tissue. Incertain embodiments, the brain tissue is in a mammal, such as a human.

The present invention provides a method of suppressing the accumulationof a target protein (e.g., aggregated and soluble Aβ) in a cell byintroducing a nucleic acid molecule described above into the cell in anamount sufficient to suppress accumulation of the target protein in thecell. In certain embodiments, the accumulation of target protein issuppressed by at least 10%. The accumulation of target protein issuppressed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%,or 99%.

Neurological Diseases

“Neurological disease” and “neurological disorder” refer to bothhereditary and sporadic conditions that are characterized by nervoussystem dysfunction, and which may be associated with atrophy of theaffected central or peripheral nervous system structures, or loss offunction without atrophy. A neurological disease or disorder thatresults in atrophy is commonly called a “neurodegenerative disease” or“neurodegenerative disorder.” Neurodegenerative diseases and disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, and repeat expansion neurodegenerativediseases, e.g., diseases associated with expansions of trinucleotiderepeats such as polyglutamine (polyQ) repeat diseases, e.g.,Huntington's disease (HD), spinocerebellar ataxia (SCA1, SCA2, SCA3,SCA6, SCAT, and SCA17), spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA). An example of a disablingneurological disorder that does not appear to result in atrophy is DYT1dystonia. The gene of interest may encode a ligand for a chemokineinvolved in the migration of a cancer cell, or a chemokine receptor.

Alzheimer's Disease

Aβ, a major protein component (4 kDa) of the senile plaque, is generatedfrom its precursor, APP, by enzymatic digestion involving β- andγ-secretase activities. The majority of Aβ fragments include Aβ₁₋₄₀ andAβ₁₋₄₂. While Aβ₁₋₄₂ is believed to be the protein initially depositedin diffuse plaques as the seed molecule for amyloid fibril formation,subsequent deposition of Aβ₁₄₀ is more closely associated with the onsetof clinical symptoms in late-onset AD, indicating that decreasing Aβconcentration represents a potentially variable therapeutic orpreventative approach. Two different approaches can be taken, one is toblock Aβ production, and a second is to clear Aβ before it can causedamage. To block Aβ production, the activity of the two differentproteases that produce Aβ from APP can be blocked: β-secretase cleavingAPP to form the amino terminal of Aβ, and γ-secretase cleaving APP toform the carboxyl terminal of AP. While inhibition of either β- orγ-secretase activity is promising and is being very actively pursued byseveral labs and pharmaceutical companies, substantial hurdles are facedsince the inhibitor must be delivered to numerous cellular locations toblock cleavage of APP. The natural functions of these enzymes arerelatively unknown so inhibition of the enzymes may result in otherdetrimental effects, and the inhibitor may block other protease activityas well causing unwanted side effects.

Cleavage of APP by β-secretase generates a ˜100 kDa soluble NH₂-terminalfragment and a 12 kDa C-terminal stub of APP (C99), which can be furthercleaved by a protease, γ-secretase. This cleavage yields two majorspecies of Aβ, one ending at residue 40 (Aβ₄₀) and the other at residue42 (Aβ₄₂). Endocytosis of APP is one route for generating Aβ; Aβ canalso be generated in the secretory pathway. Indeed, when APP wastransfected into CHO cells and then subcellularly fractionalized, Aβ₄₂was primarily detected in ER rich vesicles and Aβ₄₀ primarily inGolgi-rich vesicles.

Overexpression of β-secretase (BACE1) increases the amount of BACE1cleavage products, C99 and C89. The role of BACE1 in Aβ production invitro might explain the higher production of Aβ peptide in AD and theearly onset of Swedish familial Alzheimer's disease. It has beendemonstrated that BACE1 activity is elevated in sporadic AD brains.Studies using BACE knockout (KO) mice have demonstrated that BACE1 isthe major β-secretase for the generation of Aβ peptides and developmentof BACE1 inhibitors may be one effective approach to reduce Aβgeneration.

In addition, while γ-secretase also plays a critical role in generatingAβ, the second approach, clearing Aβ from the brain, offers theadvantage that it can avoid the problem of potentially interfering withother cell metabolic activities. Transgenic mice overexpressing mutantAPP23 or both APP23 and PS1 variants were designed to develop Aβdeposits similar to those found in AD patients. Therefore, thesetransgenic mice provide a suitable animal model to test variousstrategies to inhibit or clear Aβ deposition. Immunization of such micewith aggregated Aβ was shown to delay deposition of Aβ and also to clearAβ deposits already present in the brain. Passive immunization of thesesame mice by periodic injection of antibodies generated against Aβ wasalso shown to delay deposition of Aβ and reduce Aβ deposits that werealready present. The clearance of Aβ deposits from brain tissue in an exvivo assay was correlated with Fc receptor mediated phagocytosis.

Behavioral studies of mice that were immunized against Aβ also showedreduced memory loss and behavioral impairment. While these results arevery encouraging, there are numerous potential difficulties in applyingthis strategy to humans, including the possibility that a strongimmunization response may not be obtained or that immunization mayexacerbate inflammation in brain tissue. There is considerable evidencethat AD is an inflammatory disease and antibody mediated clearance byphagocytosis could exacerbate brain inflammation and damage. In human ADpatients, active immunization against aggregated Aβ decreased cognitivedecline and reduced neuritic pathology providing clinical evidence for arole of Aβ in AD pathology, however the study was suspended due tooccurrence of aseptic meningoencephalitis in 6% of patients.

While the inflammatory response was not directly correlated with anantibody response, the full range of classical complement proteins areupregulated in AD brains, and Aβ has been shown to bind C1q, the firstcomponents of the classical complement response. Therefore Aβ canpromote inflammation in the brain, and antibody mediated clearance ofaggregated Aβ can potentially further exacerbate this inflammation.While antibody mediated clearance represents a potentially powerfulstrategy for controlling Aβ deposition, complement activation must becarefully regulated in AD brains, stimulated enough to provide clearanceof pathogens or neurotoxic aggregates and to promote healing of neurons,but not too much that inflammation causes cognitive damage (Shen andMeri 2003).

Monoclonal antibodies to Aβ have been previously isolated and shown toinitiate clearance of aggregated Aβ, however there are severalsignificant advantages to using the scFv antibody fragments proposedhere including: 1) the scFv antibodies are derived from human cell linesand can be used for in vivo analysis and eventual treatment with lessrisk of an immunogenic response; 2) the scFv antibodies are much smallerthan monoclonals, potentially facilitating transport across the BBB; 3)peptide sequences targeting transport across the BBB can be added to therecombinant antibody constructs to further favor biodistribution to thebrain; 4) the diabody constructs can be readily modified to clear tau,prions or other potential neurodegenerative targets, and perhaps mostimportantly 5) combinations of antigenic targets and clearancemechanisms can be utilized to control inflammation.

To increase microglial clearance of AP, the inventors have developed aninnovative approach for treating AD by using bispecific recombinantantibody fragments (diabodies) that are specifically engineered toaddress the critical inflammation problem currently confronting Aβclearance as a therapeutic strategy. The bispecific antibodies willcontain two different binding activities, the first will selectivelytarget different morphologies and forms of Aβ, while the second bindingactivity will either activate different components of the classicalcomplement pathway or directly activate microglial cells to moreselectively control the inflammatory response necessary for phagocytosisof Aβ. The diabodies contain scFv fragments targeted toward eithersoluble or fibrillar Aβ in combination with scFv fragments that initiatedifferent components of the clearance mechanisms.

Aβ accumulation has been strongly correlated with AD, thereforeinhibiting AP accumulation represents a promising therapeutic strategy.The Aβ immunization trials in humans provide powerful evidence thatclearance of Aβ can be a viable therapeutic approach for treating ADproviding the inflammatory response can be controlled. Inflammatoryresponses in AD brains that are lacking in healthy brains have beenpreviously identified (Akiyama et al. 2000). While the inflammatoryresponse was not directly correlated with an antibody response, the fullrange of classical complement proteins are upregulated in AD brains(Akiyama et al. 2000), and Aβ has been shown to bind C1q, which is thefirst component of the classical complement response. Therefore Aβ canpromote inflammation in the brain, and antibody mediated clearance ofaggregated Aβ can potentially further exacerbate this inflammation.Since a delicate balance already exists in the brain between healthyclearance and destructive inflammation, a therapeutic antibody mediatedclearance strategy of Aβ must maintain this balance, as evidenced bycomplications observed in the human immunization trials (Check 2002).

Other Neurodegenerative Diseases

Beyond the tremendous potential therapeutic value for AD, the antibodyconstructs developed here represent a suitable paradigm for treatingother neurological diseases such as Parkinson's Disease, Lou Gehrig'sDisease, Huntington's Disease and spongiform encephalopathies. Theantibody constructs are designed from separate and easily substitutedfunctionalities, so the constructs can be readily modified for otherapplications. For example, prions can be targeted instead of Aβ byexchanging a prion specific scFv domain for the AP domain. Instead ofcomplement activation, a specific protease activity can be activated byswapping the complement activating scFv with a proteolytic scFv.

Antibodies and Antibody Fragments

The present invention provides a purified bi-specific ligand that bindsspecifically to a protein associated with a neurodegenerative diseaseand binds to a component of cell clearance mechanisms. As used herein,the term “antibody” includes scFv, humanized, fully human or chimericantibodies, single-chain antibodies, diabodies, and antigen-bindingfragments of antibodies that do not contain the Fc region (e.g., Fabfragments). In certain embodiments, the antibody is a human antibody ora humanized antibody. A “humanized” antibody contains only the threeCDRs (complementarity determining regions) and sometimes a few carefullyselected “framework” residues (the non-CDR portions of the variableregions) from each donor antibody variable region recombinantly linkedonto the corresponding frameworks and constant regions of a humanantibody sequence. A “fully humanized antibody” is created in ahybridoma from mice genetically engineered to have only human-derivedantibody genes or by selection from a phage-display library ofhuman-derived antibody genes.

As used herein, the term “antibody” includes a single-chain variablefragment (scFv or “nanobody”), humanized, fully human or chimericantibodies, single-chain antibodies, diabodies, and antigen-bindingfragments of antibodies (e.g., Fab fragments). A scFv is a fusionprotein of the variable region of the heavy (V_(H)) and light chains(V_(L)) of an immunoglobulin that is connected by means of a linkerpeptide. The linker is usually short, about 10-25 amino acids in length.If flexibility is important, the linker will contain a significantnumber of glycines. If solubility is important, serines or theonineswill be utilized in the linker. The linker may link the amino-terminusof the V_(H) to the carboxy-terminus of the V_(L), or the linker maylink the carboxy-terminus of the V_(H) to the amino-terminus of theV_(L). Divalent (also called bivalent) scFvs can be generated by linkingtwo scFvs. For example, a divalent scFv can be made by generating asingle peptide containing two V_(H) and two V_(L) regions.Alternatively, two peptides, each containing a single V_(H) and a singleV_(L) region can be dimerized (also called “diabodies”). Holliger etal., “Diabodies: small bivalent and bispecific antibody fragments,”PNAS, July 1993, 90:6444-6448. Bivalency allows antibodies to bind tomultimeric antigens with high avidity, and bispecificity allows thecrosslinking of two antigens.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a group of substantially homogeneous antibodies, that is,an antibody group wherein the antibodies constituting the group arehomogeneous except for naturally occurring mutants that exist in a smallamount. Monoclonal antibodies are highly specific and interact with asingle antigenic site. Furthermore, each monoclonal antibody targets asingle antigenic determinant (epitope) on an antigen, as compared tocommon polyclonal antibody preparations that typically contain variousantibodies against diverse antigenic determinants. In addition to theirspecificity, monoclonal antibodies are advantageous in that they areproduced from hybridoma cultures not contaminated with otherimmunoglobulins.

The adjective “monoclonal” indicates a characteristic of antibodiesobtained from a substantially homogeneous group of antibodies, and doesnot specify antibodies produced by a particular method. For example, amonoclonal antibody to be used in the present invention can be producedby, for example, hybridoma methods (Kohler and Milstein, Nature 256:495,1975) or recombination methods (U.S. Pat. No. 4,816,567). The monoclonalantibodies used in the present invention can be also isolated from aphage antibody library (Clackson et al., Nature 352:624-628, 1991; Markset al., J. Mol. Biol. 222:581-597, 1991). The monoclonal antibodies ofthe present invention particularly comprise “chimeric” antibodies(immunoglobulins), wherein a part of a heavy (H) chain and/or light (L)chain is derived from a specific species or a specific antibody class orsubclass, and the remaining portion of the chain is derived from anotherspecies, or another antibody class or subclass. Furthermore, mutantantibodies and antibody fragments thereof are also comprised in thepresent invention (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl.Acad. Sci. USA 81:6851-6855, 1984).

As used herein, the term “mutant antibody” refers to an antibodycomprising a variant amino acid sequence in which one or more amino acidresidues have been altered. For example, the variable region of anantibody can be modified to improve its biological properties, such asantigen binding. Such modifications can be achieved by site-directedmutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)),PCR-based mutagenesis, cassette mutagenesis, and the like. Such mutantscomprise an amino acid sequence which is at least 70% identical to theamino acid sequence of a heavy or light chain variable region of theantibody, more preferably at least 75%, even more preferably at least80%, still more preferably at least 85%, yet more preferably at least90%, and most preferably at least 95% identical. As used herein, theterm “sequence identity” is defined as the percentage of residuesidentical to those in the antibody's original amino acid sequence,determined after the sequences are aligned and gaps are appropriatelyintroduced to maximize the sequence identity as necessary.

Specifically, the identity of one nucleotide sequence or amino acidsequence to another can be determined using the algorithm BLAST, byKarlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993).Programs such as BLASTN and BLASTX were developed based on thisalgorithm (Altschul et al., J. Mol. Biol. 215: 403-410, 1990). Toanalyze nucleotide sequences according to BLASTN based on BLAST, theparameters are set, for example, as score=100 and wordlength=12. On theother hand, parameters used for the analysis of amino acid sequences byBLASTX based on BLAST include, for example, score=50 and wordlength=3.Default parameters for each program are used when using the BLAST andGapped BLAST programs. Specific techniques for such analyses are knownin the art (see the website of the National Center for BiotechnologyInformation (NCBI), Basic Local Alignment Search Tool (BLAST);http://www.ncbi.nlm.nih.gov).

Polyclonal and monoclonal antibodies can be prepared by methods known tothose skilled in the art.

In another embodiment, antibodies or antibody fragments can be isolatedfrom an antibody phage library, produced by using the technique reportedby McCafferty et al. (Nature 348:552-554 (1990)). Clackson et al.(Nature 352:624-628 (1991)) and Marks et al. (J. Mol. Biol. 222:581-597(1991)) reported on the respective isolation of mouse and humanantibodies from phage libraries. There are also reports that describethe production of high affinity (nM range) human antibodies based onchain shuffling (Marks et al., Bio/Technology 10:779-783 (1992)), andcombinatorial infection and in vivo recombination, which are methods forconstructing large-scale phage libraries (Waterhouse et al., NucleicAcids Res. 21:2265-2266 (1993)). These technologies can also be used toisolate monoclonal antibodies, instead of using conventional hybridomatechnology for monoclonal antibody production.

Antibodies to be used in the present invention can be purified by amethod appropriately selected from known methods, such as the proteinA-Sepharose method, hydroxyapatite chromatography, salting-out methodwith sulfate, ion exchange chromatography, and affinity chromatography,or by the combined use of the same.

The present invention may use recombinant antibodies, produced by geneengineering. The genes encoding the antibodies obtained by a methoddescribed above are isolated from the hybridomas. The genes are insertedinto an appropriate vector, and then introduced into a host (see, e.g.,Carl, A. K. Borrebaeck, James, W. Larrick, Therapeutic MonoclonalAntibodies, Published in the United Kingdom by Macmillan Publishers Ltd,1990). The present invention provides the nucleic acids encoding theantibodies of the present invention, and vectors comprising thesenucleic acids. Specifically, using a reverse transcriptase, cDNAsencoding the variable regions (V regions) of the antibodies aresynthesized from the mRNAs of hybridomas. After obtaining the DNAsencoding the variable regions of antibodies of interest, they areligated with DNAs encoding desired constant regions (C regions) of theantibodies, and the resulting DNA constructs are inserted intoexpression vectors. Alternatively, the DNAs encoding the variableregions of the antibodies may be inserted into expression vectorscomprising the DNAs of the antibody C regions. These are inserted intoexpression vectors so that the genes are expressed under the regulationof an expression regulatory region, for example, an enhancer andpromoter. Then, host cells are transformed with the expression vectorsto express the antibodies. The present invention provides cellsexpressing antibodies of the present invention. The cells expressingantibodies of the present invention include cells and hybridomastransformed with a gene of such an antibody.

The antibodies of the present invention also include antibodies whichcomprise complementarity-determining regions (CDRs), or regionsfunctionally equivalent to CDRs. The term “functionally equivalent”refers to comprising amino acid sequences similar to the amino acidsequences of CDRs of any of the monoclonal antibodies isolated in theExamples. The term “CDR” refers to a region in an antibody variableregion (also called “V region”), and determines the specificity ofantigen binding. The H chain and L chain each have three CDRs,designated from the N terminus as CDR1, CDR2, and CDR3. There are fourregions flanking these CDRs: these regions are referred to as“framework,” and their amino acid sequences are highly conserved. TheCDRs can be transplanted into other antibodies, and thus a recombinantantibody can be prepared by combining CDRs with the framework of adesired antibody. One or more amino acids of a CDR can be modifiedwithout losing the ability to bind to its antigen. For example, one ormore amino acids in a CDR can be substituted, deleted, and/or added.

In certain embodiments, an amino acid residue is mutated into one thatallows the properties of the amino acid side-chain to be conserved.Examples of the properties of amino acid side chains comprise:hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic aminoacids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising thefollowing side chains: aliphatic side-chains (G, A, V, L, I, P);hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containingside-chains (C, M); carboxylic acid- and amide-containing side-chains(D, N, E, Q); base-containing side-chains (R, K, H); andaromatic-containing side-chains (H, F, Y, W). The letters withinparenthesis indicate the one-letter amino acid codes. Amino acidsubstitutions within each group are called conservative substitutions.It is well known that a polypeptide comprising a modified amino acidsequence in which one or more amino acid residues is deleted, added,and/or substituted can retain the original biological activity (Mark D.F. et al., Proc. Natl. Acad. Sci. U.S.A. 81:5662-5666 (1984); Zoller M.J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. etal., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl.Acad. Sci. U.S.A. 79: 6409-6413 (1982)). The number of mutated aminoacids is not limited, but in general, the number falls within 40% ofamino acids of each CDR, and preferably within 35%, and still morepreferably within 30% (e.g., within 25%). The identity of amino acidsequences can be determined as described herein.

In the present invention, recombinant antibodies artificially modifiedto reduce heterologous antigenicity against humans can be used. Examplesinclude chimeric antibodies and humanized antibodies. These modifiedantibodies can be produced using known methods. A chimeric antibodyincludes an antibody comprising variable and constant regions of speciesthat are different to each other, for example, an antibody comprisingthe antibody heavy chain and light chain variable regions of a nonhumanmammal such as a mouse, and the antibody heavy chain and light chainconstant regions of a human. Such an antibody can be obtained by (1)ligating a DNA encoding a variable region of a mouse antibody to a DNAencoding a constant region of a human antibody; (2) incorporating thisinto an expression vector; and (3) introducing the vector into a hostfor production of the antibody.

A humanized antibody, which is also called a reshaped human antibody, isobtained by substituting an H or L chain complementarity determiningregion (CDR) of an antibody of a nonhuman mammal such as a mouse, withthe CDR of a human antibody. Conventional genetic recombinationtechniques for the preparation of such antibodies are known (see, forexample, Jones et al., Nature 321: 522-525 (1986); Reichmann et al.,Nature 332: 323-329 (1988); Presta Curr. Op. Struct. Biol. 2: 593-596(1992)). Specifically, a DNA sequence designed to ligate a CDR of amouse antibody with the framework regions (FRs) of a human antibody issynthesized by PCR, using several oligonucleotides constructed tocomprise overlapping portions at their ends. A humanized antibody can beobtained by (1) ligating the resulting DNA to a DNA that encodes a humanantibody constant region; (2) incorporating this into an expressionvector; and (3) transfecting the vector into a host to produce theantibody (see, European Patent Application No. EP 239,400, andInternational Patent Application No. WO 96/02576). Human antibody FRsthat are ligated via the CDR are selected where the CDR forms afavorable antigen-binding site. The humanized antibody may compriseadditional amino acid residue(s) that are not included in the CDRsintroduced into the recipient antibody, nor in the framework sequences.Such amino acid residues are usually introduced to more accuratelyoptimize the antibody's ability to recognize and bind to an antigen. Forexample, as necessary, amino acids in the framework region of anantibody variable region may be substituted such that the CDR of areshaped human antibody forms an appropriate antigen-binding site (Sato,K. et al., Cancer Res. (1993) 53, 851-856).

The isotypes of the antibodies of the present invention are not limited.The isotypes include, for example, IgG (IgG1, IgG2, IgG3, and IgG4),IgM, IgA (IgA1 and IgA2), IgD, and IgE. The antibodies of the presentinvention may also be antibody fragments comprising a portionresponsible for antigen binding, or a modified fragment thereof. Theterm “antibody fragment” refers to a portion of a full-length antibody,and generally to a fragment comprising an antigen-binding domain or avariable region. Such antibody fragments include, for example, Fab,F(ab′)₂, Fv, single-chain Fv (scFv) which comprises a heavy chain Fv anda light chain Fv coupled together with an appropriate linker, diabody(diabodies), linear antibodies, and multispecific antibodies preparedfrom antibody fragments. Previously, antibody fragments were produced bydigesting natural antibodies with a protease; currently, methods forexpressing them as recombinant antibodies using genetic engineeringtechniques are also known (see Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992); Brennan et al., Science229:81 (1985); Co, M. S. et al., J. Immunol., 1994, 152, 2968-2976;Better, M. & Horwitz, A. H., Methods in Enzymology, 1989, 178, 476-496,Academic Press, Inc.; Plueckthun, A. & Skerra, A., Methods inEnzymology, 1989, 178, 476-496, Academic Press, Inc.; Lamoyi, E.,Methods in Enzymology, 1989, 121, 663-669; Bird, R. E. et al., TIBTECH,1991, 9, 132-137).

An “Fv” fragment is the smallest antibody fragment, and contains acomplete antigen recognition site and a binding site. This region is adimer (V_(H)—V_(L) dimer) wherein the variable regions of each of theheavy chain and light chain are strongly connected by a noncovalentbond. The three CDRs of each of the variable regions interact with eachother to form an antigen-binding site on the surface of the V_(H)-V_(L)dimer. In other words, a total of six CDRs from the heavy and lightchains function together as an antibody's antigen-binding site. However,a variable region (or a half Fv, which contains only threeantigen-specific CDRS) alone is also known to be able to recognize andbind to an antigen, although its affinity is lower than the affinity ofthe entire binding site. Thus, a preferred antibody fragment of thepresent invention is an Fv fragment, but is not limited thereto. Such anantibody fragment may be a polypeptide which comprises an antibodyfragment of heavy or light chain CDRs which are conserved, and which canrecognize and bind its antigen.

A Fab fragment (also referred to as F(ab)) also contains a light chainconstant region and heavy chain constant region (CH1). For example,papain digestion of an antibody produces the two kinds of fragments: anantigen-binding fragment, called a Fab fragment, containing the variableregions of a heavy chain and light chain, which serve as a singleantigen-binding domain; and the remaining portion, which is called an“Fc” because it is readily crystallized. A Fab′ fragment is differentfrom a Fab fragment in that a Fab′ fragment also has several residuesderived from the carboxyl terminus of a heavy chain CH1 region, whichcontains one or more cysteine residues from the hinge region of anantibody. A Fab′ fragment is, however, structurally equivalent to Fab inthat both are antigen-binding fragments which comprise the variableregions of a heavy chain and light chain, which serve as a singleantigen-binding domain. Herein, an antigen-binding fragment comprisingthe variable regions of a heavy chain and light chain which serve as asingle antigen-binding domain, and which is equivalent to that obtainedby papain digestion, is referred to as a “Fab-like antibody,” even whenit is not identical to an antibody fragment produced by proteasedigestion. Fab′-SH is Fab′ with one or more cysteine residues havingfree thiol groups in its constant region. A F(ab′) fragment is producedby cleaving the disulfide bond between the cysteine residues in thehinge region of F(ab′)₂. Other chemically crosslinked antibody fragmentsare also known to those skilled in the art. Pepsin digestion of anantibody yields two fragments; one is a F(ab′)₂ fragment which comprisestwo antigen-binding domains and can cross-react with antigens, and theother is the remaining fragment (referred to as pFc′). Herein, anantibody fragment equivalent to that obtained by pepsin digestion isreferred to as a “F(ab′)₂-like antibody” when it comprises twoantigen-binding domains and can cross-react with antigens. Such antibodyfragments can also be produced, for example, by genetic engineering.Such antibody fragments can also be isolated, for example, from theantibody phage library described above. Alternatively, F(ab′)₂-SHfragments can be recovered directly from hosts, such as E. coli, andthen allowed to form F(ab′)₂ fragments by chemical crosslinking (Carteret al., Bio/Technology 10:163-167 (1992)). In an alternative method,F(ab′)₂ fragments can be isolated directly from a culture of recombinanthosts.

The term “diabody (Db)” refers to a bivalent antibody fragmentconstructed by gene fusion (for example, P. Holliger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO 93/11161). Ingeneral, a diabody is a dimer of two polypeptide chains. In the each ofthe polypeptide chains, a light chain variable region (V_(L)) and aheavy chain variable region (V_(H)) in an identical chain are connectedvia a short linker, for example, a linker of about five residues, sothat they cannot bind together. Because the linker between the two istoo short, the V_(L) and V_(H) in the same polypeptide chain cannot forma single chain V region fragment, but instead form a dimer. Thus, adiabody has two antigen-binding domains. When the V_(L) and V_(H)regions against the two types of antigens (a and b) are combined to formV_(La)-V_(Hb) and V_(Lb)-V_(Ha) via a linker of about five residues, andthen co-expressed, they are secreted as bispecific Dbs. The antibodiesof the present invention may be such Dbs.

A single-chain antibody (also referred to as “scFv”) can be prepared bylinking a heavy chain V region and a light chain V region of an antibody(for a review of scFv see Pluckthun “The Pharmacology of MonoclonalAntibodies” Vol. 113, eds. Rosenburg and Moore, Springer Verlag, N.Y.,pp. 269-315 (1994)). Methods for preparing single-chain antibodies areknown in the art (see, for example, U.S. Pat. Nos. 4,946,778; 5,260,203;5,091,513; and 5,455,030). In such scFvs, the heavy chain V region andthe light chain V region are linked together via a linker, preferably, apolypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A,1988, 85, 5879-5883). The heavy chain V region and the light chain Vregion in a scFv may be derived from the same antibody, or fromdifferent antibodies. The peptide linker used to ligate the V regionsmay be any single-chain peptide consisting of 12 to 19 residues. A DNAencoding a scFv can be amplified by PCR using, as a template, either theentire DNA, or a partial DNA encoding a desired amino acid sequence,selected from a DNA encoding the heavy chain or the V region of theheavy chain of the above antibody, and a DNA encoding the light chain orthe V region of the light chain of the above antibody; and using aprimer pair that defines the two ends. Further amplification can besubsequently conducted using a combination of the DNA encoding thepeptide linker portion, and the primer pair that defines both ends ofthe DNA to be ligated to the heavy and light chain respectively. Afterconstructing DNAs encoding scFvs, conventional methods can be used toobtain expression vectors comprising these DNAs, and hosts transformedby these expression vectors. Furthermore, scFvs can be obtainedaccording to conventional methods using the resulting hosts. Theseantibody fragments can be produced in hosts by obtaining genes thatencode the antibody fragments and expressing these as outlined above.Antibodies bound to various types of molecules, such as polyethyleneglycols (PEGs), may be used as modified antibodies. Methods formodifying antibodies are already established in the art. The term“antibody” in the present invention also encompasses the above-describedantibodies.

The antibodies obtained can be purified to homogeneity. The antibodiescan be isolated and purified by a method routinely used to isolate andpurify proteins. The antibodies can be isolated and purified by thecombined use of one or more methods appropriately selected from columnchromatography, filtration, ultrafiltration, salting out, dialysis,preparative polyacrylamide gel electrophoresis, and isoelectro-focusing,for example (Strategies for Protein Purification and Characterization: ALaboratory Course Manual, Daniel R. Marshak et al. eds., Cold SpringHarbor Laboratory Press (1996); Antibodies: A Laboratory Manual. EdHarlow and David Lane, Cold Spring Harbor Laboratory, 1988). Suchmethods are not limited to those listed above. Chromatographic methodsinclude affinity chromatography, ion exchange chromatography,hydrophobic chromatography, gel filtration, reverse-phasechromatography, and adsorption chromatography. These chromatographicmethods can be practiced using liquid phase chromatography, such as HPLCand FPLC. Columns to be used in affinity chromatography include proteinA columns and protein G columns. For example, protein A columns includeHyper D, POROS, and Sepharose F. F. (Pharmacia). Antibodies can also bepurified by utilizing antigen binding, using carriers on which antigenshave been immobilized.

The antibodies of the present invention can be formulated according tostandard methods (see, for example, Remington's Pharmaceutical Science,latest edition, Mark Publishing Company, Easton, U.S.A), and maycomprise pharmaceutically acceptable carriers and/or additives. Thepresent invention relates to compositions (including reagents andpharmaceuticals) comprising the antibodies of the invention, andpharmaceutically acceptable carriers and/or additives. Exemplarycarriers include surfactants (for example, PEG and Tween), excipients,antioxidants (for example, ascorbic acid), coloring agents, flavoringagents, preservatives, stabilizers, buffering agents (for example,phosphoric acid, citric acid, and other organic acids), chelating agents(for example, EDTA), suspending agents, isotonizing agents, binders,disintegrators, lubricants, fluidity promoters, and corrigents. However,the carriers that may be employed in the present invention are notlimited to this list. In fact, other commonly used carriers can beappropriately employed: light anhydrous silicic acid, lactose,crystalline cellulose, mannitol, starch, carmelose calcium, carmelosesodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose,polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin,medium chain fatty acid triglyceride, polyoxyethylene hydrogenatedcastor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganicsalt, and so on. The composition may also comprise otherlow-molecular-weight polypeptides, proteins such as serum albumin,gelatin, and immunoglobulin, and amino acids such as glycine, glutamine,asparagine, arginine, and lysine. When the composition is prepared as anaqueous solution for injection, it can comprise an isotonic solutioncomprising, for example, physiological saline, dextrose, and otheradjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol,and sodium chloride, which can also contain an appropriate solubilizingagent, for example, alcohol (for example, ethanol), polyalcohol (forexample, propylene glycol and PEG), and non-ionic detergent (polysorbate80 and HCO-50).

If necessary, antibodies of the present invention may be encapsulated inmicrocapsules (microcapsules made of hydroxycellulose, gelatin,polymethylmethacrylate, and the like), and made into components ofcolloidal drug delivery systems (liposomes, albumin microspheres,microemulsions, nano-particles, and nano-capsules) (for example, see“Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980)).Moreover, methods for making sustained-release drugs are known, andthese can be applied for the antibodies of the present invention (Langeret al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech.12: 98-105 (1982); U.S. Pat. No. 3,773,919; EP Patent Application No.58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP: 133,988).

Nucleic Acid Molecules Encoding Antibodies

The present invention further provides nucleic acid sequences thatencode the antibodies described above.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991);Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell.Probes, 8:91 (1994). A “nucleic acid fragment” is a fraction of a givennucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority oforganisms is the genetic material while ribonucleic acid (RNA) isinvolved in the transfer of information contained within DNA intoproteins. The term “nucleotide sequence” refers to a polymer of DNA orRNA that can be single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The terms “nucleic acid,”“nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequenceor segment,” or “polynucleotide” may also be used interchangeably withgene, cDNA, DNA and RNA encoded by a gene.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that exists apart from itsnative environment and is therefore not a product of nature. An isolatedDNA molecule or polypeptide may exist in a purified form or may exist ina non-native environment such as, for example, a transgenic host cell.For example, an “isolated” or “purified” nucleic acid molecule orprotein, or biologically active portion thereof, is substantially freeof other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. In one embodiment, an“isolated” nucleic acid is free of sequences that naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. A protein that is substantially free ofcellular material includes preparations of protein or polypeptide havingless than about 30%, 20%, 10%, 5%, (by dry weight) of contaminatingprotein. When the protein of the invention, or biologically activeportion thereof, is recombinantly produced, preferably culture mediumrepresents less than about 30%, 20%, 10%, or 5% (by dry weight) ofchemical precursors or non-protein-of-interest chemicals. Fragments andvariants of the disclosed nucleotide sequences and proteins orpartial-length proteins encoded thereby are also encompassed by thepresent invention. By “fragment” or “portion” is meant a full length orless than full length of the nucleotide sequence encoding, or the aminoacid sequence of, a polypeptide or protein.

“Naturally occurring” is used to describe an object that can be found innature as distinct from being artificially produced. For example, aprotein or nucleotide sequence present in an organism (including avirus), which can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory, is naturallyoccurring.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis that encode the native protein, as wellas those that encode a polypeptide having amino acid substitutions.Generally, nucleotide sequence variants of the invention will have atleast 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%,e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to98%, sequence identity to the native (endogenous) nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (3^(rd) edition, 2001).

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” each refer to a sequence that originatesfrom a source foreign to the particular host cell or, if from the samesource, is modified from its original form. Thus, a heterologous gene ina host cell includes a gene that is endogenous to the particular hostcell but has been modified. The terms also include non-naturallyoccurring multiple copies of a naturally occurring DNA sequence. Thus,the terms refer to a DNA segment that is foreign or heterologous to thecell, or homologous to the cell but in a position within the host cellnucleic acid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

A “homologous” DNA sequence is a DNA sequence that is naturallyassociated with a host cell into which it is introduced.

“Wild-type” refers to the normal gene, or organism found in naturewithout any known mutation.

“Genome” refers to the complete genetic material of an organism.

A “vector” is defined to include, inter alia, any plasmid, cosmid, phageor binary vector in double or single stranded linear or circular formwhich may or may not be self transmissible or mobilizable, and which cantransform prokaryotic or eukaryotic host either by integration into thecellular genome or exist extrachromosomally (e.g., autonomousreplicating plasmid with an origin of replication).

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one that isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter that initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

Such expression cassettes will comprise the transcriptional initiationregion of the invention linked to a nucleotide sequence of interest.Such an expression cassette is provided with a plurality of restrictionsites for insertion of the gene of interest to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences that may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters. However, some suitableregulatory sequences useful in the present invention will include, butare not limited to constitutive promoters, tissue-specific promoters,development-specific promoters, inducible promoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., Mol. Biotech., 3:225 (1995).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may also contain DNAsequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition+1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence (Le., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation in a cell ofan endogenous gene, transgene, as well as the transcription and stableaccumulation of sense (mRNA) or functional RNA. In the case of antisenseconstructs, expression may refer to the transcription of the antisenseDNA only. Expression may also refer to the production of protein.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples oftranscription stop fragments are known to the art.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

The terms “cis-acting sequence” and “cis-acting element” refer to DNA orRNA sequences whose functions require them to be on the same molecule.

The terms “trans-acting sequence” and “trans-acting element” refer toDNA or RNA sequences whose function does not require them to be on thesame molecule.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, CABIOS, 4:11 (1988); the local homology algorithmof Smith et al., Adv. Appl. Math., 2:482 (1981); the homology alignmentalgorithm of Needleman and Wunsch, J M B, 48:443 (1970); thesearch-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85:2444 (1988); the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 87:2264 (1990), modified as in Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.,Gene, 73:237 (1988); Higgins et al., CABIOS, 5:151 (1989); Corpet etal., Nucl. Acids Res., 16:10881 (1988); Huang et al., CABIOS, 8:155(1992); and Pearson et al., Meth. Mol. Biol., 24:307 (1994). The ALIGNprogram is based on the algorithm of Myers and Miller, supra. The BLASTprograms of Altschul et al., JMB, 215:403 (1990); Nucl. Acids Res.,25:3389 (1990), are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (available on the worldwide web at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al., NucleicAcids Res. 25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al., supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of100, M=5, N=−4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3, anexpectation (E) of 10, and the BLOSUM62 scoring matrix. See the worldwide web at ncbi.nlm.nih.gov. Alignment may also be performed manuallyby visual inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%,and at least 95%, 96%, 97%, 98°/or 99% sequence identity, compared to areference sequence using one of the alignment programs described usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 70%, at least 80%,90%, at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%,97%, 98% or 99%, sequence identity to the reference sequence over aspecified comparison window. Optimal alignment is conducted using thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 (1970). An indication that two peptide sequences aresubstantially identical is that one peptide is immunologically reactivewith antibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138:267 (1984); T_(m) 81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. T_(m) is reduced by about 1° C. foreach 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the T_(m); moderately stringent conditionscan utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lowerthan the T_(m); low stringency conditions can utilize a hybridizationand/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the T_(m). Usingthe equation, hybridization and wash compositions, and desiredtemperature, those of ordinary skill will understand that variations inthe stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a temperatureof less than 45° C. (aqueous solution) or 32° C. (formamide solution),it is preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology Hybridization with Nucleic Acid Probes, part Ichapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays” Elsevier, New York (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the T_(m) for the specific sequence at a defined ionicstrength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long probes (e.g., >50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Nucleic acids that do not hybridize to eachother under stringent conditions are still substantially identical ifthe proteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultsform, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the polypeptides canbe prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Kunkel etal., Meth. Enzymol., 154:367 (1987); U.S. Pat. No. 4,873,192; Walker andGaastra, Techniques in Mol. Biol. (MacMillan Publishing Co. (1983), andthe references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., Atlas of ProteinSequence and Structure (Natl. Biomed. Res. Found. 1978). Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, are preferred.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, thepolypeptides of the invention encompass naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired activity. The deletions, insertions, andsubstitutions of the polypeptide sequence encompassed herein are notexpected to produce radical changes in the characteristics of thepolypeptide. However, when it is difficult to predict the exact effectof the substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence are also “conservatively modified variations.”

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.

“Transformed,” “transgenic,” and “recombinant” refer to a host cell ororganism into which a heterologous nucleic acid molecule has beenintroduced. The nucleic acid molecule can be stably integrated into thegenome generally known in the art and are disclosed in Sambrook andRussell, supra. See also Innis et al., PCR Protocols, Academic Press(1995); and Gelfand, PCR Strategies, Academic Press (1995); and Innisand Gelfand, PCR Methods Manual, Academic Press (1999). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially mismatchedprimers, and the like. For example, “transformed,” “transformant,” and“transgenic” cells have been through the transformation process andcontain a foreign gene integrated into their chromosome. The term“untransformed” refers to normal cells that have not been through thetransformation process.

A “transgenic” organism is an organism having one or more cells thatcontain an expression vector.

By “portion” or “fragment,” as it relates to a nucleic acid molecule,sequence or segment of the invention, when it is linked to othersequences for expression, is meant a sequence having at least 80nucleotides, more preferably at least 150 nucleotides, and still morepreferably at least 400 nucleotides. If not employed for expressing, a“portion” or “fragment” means at least 9, preferably 12, more preferably15, even more preferably at least 20, consecutive nucleotides, e.g.,probes and primers (oligonucleotides), corresponding to the nucleotidesequence of the nucleic acid molecules of the invention.

As used herein, the term “therapeutic agent” refers to any agent ormaterial that has a beneficial effect on the mammalian recipient. Thus,“therapeutic agent” embraces both therapeutic and prophylactic moleculeshaving nucleic acid or protein components.

“Treating” as used herein refers to ameliorating at least one symptomof, curing and/or preventing the development of a given disease orcondition.

Formulations and Methods of Administration

For in vivo use, a therapeutic agent as described herein is generallyincorporated into a pharmaceutical composition prior to administration.Within such compositions, one or more therapeutic compounds as describedherein are present as active ingredient(s) (i.e., are present at levelssufficient to provide a statistically significant effect on the symptomsof cystic fibrosis, as measured using a representative assay). Apharmaceutical composition comprises one or more such compounds incombination with any pharmaceutically acceptable carrier(s) known tothose skilled in the art to be suitable for the particular mode ofadministration. In addition, other pharmaceutically active ingredients(including other therapeutic agents) may, but need not, be presentwithin the composition.

The term “therapeutically effective amount,” in reference to treating adisease state/condition, refers to an amount of a compound either aloneor as contained in a pharmaceutical composition that is capable ofhaving any detectable, positive effect on any symptom, aspect, orcharacteristics of a disease state/condition when administered as asingle dose or in multiple doses. Such effect need not be absolute to bebeneficial.

The terms “treat,” “treating” and “treatment” as used herein includeadministering a compound prior to the onset of clinical symptoms of adisease state/condition so as to prevent any symptom, as well asadministering a compound after the onset of clinical symptoms of adisease state/condition so as to reduce or eliminate any symptom, aspector characteristic of the disease state/condition. Such treating need notbe absolute to be useful.

In certain embodiments, the present therapeutic agent may besystemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the active compound may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of a given unit dosage form. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts may be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient that are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions that can be used todeliver the compounds of the present invention to the skin are known tothe art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157)and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of the present inventionin a liquid composition, such as a lotion, will be from about 0.1-25wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

EXAMPLE 1

Transgenic mice have been developed that overexpress a mutant form ofAPP and develop neuropathological Aβ plaques, show delayed depositionand even clearance of these plaques when immunized with Aβ. Theseresults were extended to show that the same protection against Aβdeposition could also be achieved by passive immunization whereantibodies raised against Aβ were periodically injected into mice (Bardet al. 2000). These studies raise the possibility that a carefullydesigned passive immunization approach can be used to clear Aβ in humanswhile controlling the delicate balance between clearance andinflammation. In order to understand how antibody mediated phagocytosiscan be more effectively employed for the treatment of AD, below is abrief outline of a few of the critical components of the antibodymediated complement response system.

The complement system consists of over 20 different proteins thatinteract in a highly orchestrated series of enzymatic reactions whichgenerate products that facilitate and amplify antigen clearance. Thecomplement system can be activated by two different pathways, theclassical or alternative pathways, both of which end up activating thesame complexes. The classical pathway is initiated by antibody bindingwhile the alternative pathway is generally initiated by foreigncell-surface components. The classical pathway is therefore the pathwayof interest in the antibody mediated clearance of Aβ. The classicalpathway is initiated by the formation of an antibody-antigen complex.Certain antibodies including IgM, IgG1, IgG2 and IgG3 are capable ofactivating the classical complement response upon antigen binding. Whenthese antibodies bind their target antigen, the resulting complexinduces a conformational change in the antibody Fc region. Theconformational change in the Fc region exposes a binding site for thefirst component of the classical complement system, the macromolecularprotein complex C1. C1 exists in serum as a complex containing one C1qmolecule and two molecules each of C1r and C1s. Two C1-antibodyinteractions are generally required to form a stable complex. Since IgGhas only one binding site for C1q, two IgG molecules bound on theantigenic surface within a distance of 30-40 nm are required forcomplement activation. IgM however can adapt a pentameric structure whenbound to antigen and therefore a single molecule of IgM can stably bindC1 and initiate a complement response. In order to phagocytose eithersoluble or aggregated Aβ deposits, the antibodies must bind to thesurface exposing the C1q binding site on the Fc portion of the antibody.Two C1q binding sites must be located in close enough proximity to forma stable antibody-C1 complex, which in turn initiates the complementresponse cascade ending with C3b and C5b bound to the antigenic surface.Phagocytic cells then bind C3b through CR1 receptors initiatingphagocytosis and also forming C3bi, which in turn binds CR3 receptorsfurther promoting phagocytosis.

Monoclonal antibody preparations may have difficulty in clearing Aβ,particularly soluble Aβ, since formation of a stable antibody C1 complexrequires two adjacent Fc sites. Bispecific recombinant antibodies aremade to both soluble and aggregated Aβ, which can activate antibodyclearance through different mechanisms. The inventors have developedscFv fragments directed toward different components of the complementresponse cascade. One scFv fragment binds C1q in a manner that initiatesclearance. A diabody construct containing an scFv binding hen egglysozyme (HEL) and the scFv binding C1 q was shown to promotephagocytosis of red blood cells coated with HEL. A second scFv fragmentbinds serum Ig, binding both IgM and IgG. Diabody constructs containingan scFv targeting HEL along with the scFv binding serum Ig also promotedphagocytosis of red blood cells coated with HEL. In addition, scFvsbinding different receptors that activate inflammatory or phagocyticresponses in leukocytes (anti-CD16; and anti-CD64) are available toinduce a response with the functionally related microglial cells.Further, new scFvs that directly activate microglial cells are isolated.Since ex vivo assays are predictive of in vivo clearance of Aβ depositsin mice (Bard et al. 2000), the ex vivo assay is used as a method toscreen various combinations of bispecific antibodies targeting differentmorphologies of Aβ and activating different clearance mechanisms.

Generation of APP23/C1q^(−/−) Mice.

Alzheimer-mouse model, APP23, overproduce Aβ and develop significantamyloid deposits. Brain homogenates from APP23 mice by ELISAs andimmunoprecipitation blotting for Aβ levels were analyzed. Increased Aβlevels were observed in APP mice with age using a Sandwich ELISA (FIGS.1A and 1B). Further, Aβ40 and Aβ42 were visualized by using a ureaSDS/PAGE gel. Briefly, mouse brain homogenates were dissolved in formicacid buffer and immunoprecipitated with rabbit anti-Aβ polyclonalantibody and sample was separated on a 12% Urea gel. Aβ40 and Aβ42 weredetected by the 6E10 antibody. APP23 mice were crossed with micedepleted of C1q (C1 q^(−/−)) to generate APP23/C1q^(−/−) mice.

Construction of Aβ C1q Diabody.

In order to develop a therapeutic to clear Aβ in vivo, scFv fragmentsthat bind both soluble and fibrillar Aβ are combined with scFv fragmentsthat activate different components of the complement cascade or thatbind to different receptors on leukocytes or that directly activatemicroglial cells. Each scFv consists of a light chain and heavy chainfragment connected with a short linker sequence into a single peptide.In certain embodiments, diabodies are constructed by genetically linkingtwo scFvs in series using a long flexible (Gly₄-Ser)₃ linker (SEQ ID NO:1). In certain embodiments, the diabodies are designed to also contain apoly-His tail to facilitate purification using metal ion affinitychromatography.

Two scFv diabody clones that have already been shown to activate thecomplement response and clear their target compounds throughphagocytosis have been obtained, one binds serum immunoglobulin and theother complement component C1q. The inventors constructed a H1v2-C1qdiabody and have purified fully functional bispecific diabody protein.In other embodiments, diabody molecules specific for Aβ42, oligomericand fibrillar Aβ are constructed (Example 2). In addition, scFvs thatbind to CD16 and CD64, both of which activate clearance by phagocyticcells, are available, and both have proven to be effective as bispecificdiabody constructs for targeting and killing tumor cells (Curnow 1997;McCall et al. 1999).

To generate a diabody containing scFvs to both β-amyloid and C1q, theinventors cloned the H1v2 scFv gene isolated against β-amyloiddownstream to the scFv against C1q. A (Gly₄Ser)₃ peptide linker (SEQ IDNO: 1) was used to connect the carboxyl terminus of H1v2 with the aminoterminus of the anti-C1q scFv. The resulting diabody has two domains,linked by a (Gly₄Ser)₃ peptide linker (SEQ ID NO: 1) (FIG. 2). Thediabody gene is under control of lac promoter and is in frame with gIIIsignal sequence at the 5′ terminal. To facilitate detection and recoveryof the diabody, a c-myc tag and a (His)₆ tag (SEQ ID NO: 2) wereinserted in frame at the 3′ terminal of the bispecific scFv. The gIIIsignal sequence directs the polypeptide into the periplasmic space,where it can fold correctly in a soluble form. The c-myc tag is used toanalyze the expression level of the bispecific scFv, and (His)₆ tag (SEQID NO: 2) can be used to purified the bispecific scFv protein.

Protein Purification.

After induction with IPTG, the soluble periplasmic content of the E.coli culture was purified by IMAC (immobilized metal affinitychromatography). SDS-PAGE analysis of peak fractions eluted using 50 mMimidazole indicated one fraction containing a single strong band of size60 kDa, the size expected for the diabody. Western blot analysis of thissample using a secondary antibody to the myc-tag site indicated a singlereactive band, with the expected size of 60 kD.

Antigen Binding Activity of the Purified Bispecific Antibody Fragment.

A biosensor sandwich assay utilizing immobilized Aβ on the biosensorchip surface was used to demonstrate that both Aβ and C1q bindingactivities were present. After addition of the purified diabody sample,the signal increased by around 100 RU as expected due to binding of thediabody to Aβ (FIG. 3, phase 1). After a rinse step to dissociatenon-specifically bound diabody, the second antigen, C1q, was injectedover the chip surface, resulting in a further 200 RU increase in theobserved signal corresponding to C1q binding to the diabody (FIG. 3Phase 2). These results demonstrate that both binding activities arefunctional in the diabody construct.

Isolating Antibodies that Activate Microglial Cells.

While scFvs that activate leukocytes are available, the scFvs may notbind similar receptors microglial cells, which are much less wellcharacterized. In order to ensure that the scFvs can directly activatemicroglial cells, a phage-display library is panned for members thatactivate microglial cells. There are two different approaches can beused to isolate scFvs that activate microglial cells: the first issimilar to conventional biopanning using microtitre wells, the second isto use a cell sorting flow cytometer (FACS).

In the first approach, microglial cells are incubated with aliquots fromthe antibody library. Non-binding phage is eluted, and bound phagerecovered by infection with E. coli as routinely done in otherbiopanning protocols. After several rounds of panning in this mannersingle antibody clones are assayed for microglial activation. Thoseclones that activate microglial cells are used in diabody constructs forex-vivo Aβ targeting and clearance assays.

Alternatively, phage are identified that activate microglial cells bydirectly isolating activated microglial cells and recovering boundphage. FACS is used to sort out activated microglial cells containingphage displayed antibody bound to a cell receptor site. Specifically,the microglial cells are cultured with phage-displayed antibodies forvarious time periods, after which unbound phage is washed away. Thecells are then be stained with fluorescence-labeled anti-phage antibody.Since the activated microglial cells are much bigger than resting cells,activated microglial cells can be isolated based on both fluorescenceintensity and forward scatter/size scatter. Recovered scFv fragments aretested for ability to activate microglial cells, and then used fordiabody constructs and ex-vivo assays.

Ex Vivo Aβ Clearance Assays Using Microglial Cells.

Since in vivo immunization studies are time consuming and expensive, anassay to select the most promising antibody constructs represent avaluable screening tool. An ex vivo assay for Aβ clearance has beenshown to reliably predict results obtained in in vivo Aβ clearancestudies (Bard et al. 2000). Since initiating the complement cascaderequires binding of two adjacent Fc regions within 30-40 nm of eachother, in certain embodiments two different scFv diabodies containingscFvs that recognize two different regions of the Aβ molecule areutilized. However, since Aβ can also bind C1q by itself, in certainembodiments a second binding site at the scFv is sufficient to initiateclearance. In certain embodiments, the scFv directed toward serum Iginitiates clearance of soluble Aβ alone since it is capable of bindingIgM as well as IgG. Since only one molecule of IgM can multiply bindC1q, forming a stable complex, in certain embodiments, a single diabodytargeting soluble Aβ and serum Ig initiates phagocytosis.

a) Isolation and Characterization of Microglia.

Microglial cells are obtained from mouse brain tissues and are preparedand isolated according to a previously described method (Lue et al.1996). Briefly, frontal cortex samples are removed at autopsy underaseptic conditions, then quickly immersed in ice cold Hank's balancedsalt solution (HBSS) (Irvine Scientific, CA). Enzymatic dissociation isprocessed as described elsewhere (Kim et al. 1983). Samples are washedwith several changes of HBSS, after which all visible connective tissuesand blood vessels are removed. The tissues are minced and incubated witha Ca²⁺ and Mg²⁺ free HBSS solution with 2.5% trypsin (Life Technologies,MD) and 2 mg/mL deoxyribonuclease I (Amersham, Ill.), then incubated ina shaking water bath at 37 C for 30 min. and 150 rpm. After theincubation, 2 mL of fetal bovine serum is added to stop the enzymaticdigestion. The digested sample is triturated and centrifuged at 1500 rpmfor 30 min. The pellets are resuspended in 40 mL HBSS and filteredthrough 130 μm, 100 μm and 200 μm mesh. The filtrate is subsequentlyspun at 15,000 rpm in 100% Percoll (Sigma) for 30 min at 3 C. The viablecell layer (middle layer) is transferred to 50 mL centrifuge tubes andwashed twice with HBSS. A third wash is performed in growth mediumconsisting of DMEM (Life Technologies) with high glucose (25 mM), 2%HEPES, 1% sodium pyruvate, 10% fetal bovine serum (FBS), and 0.1%gentamicin (Sigma). After the third wash, cells are resuspended ingrowth medium and plated in 24 well plates (Corning, N.Y.) coated withpoly-D-lysine (Sigma) at a density of about 1.0×10⁶ cells per well.Cells are incubated with the growth medium at 37 C in 5% CO₂ for 18-24hours when microglia cells will adhere to the culture ware surfaces. Thenon-adherent astrocytes are removed. The microglial cells are thencultured in the growth medium with weekly medium change, and cells areused two weeks after the initial plating for studies.

b) Immunofluorescence Phagocytosis Assay with Treatments of BispecificAntibodies.

Microglia phagocytic activity is quantified by measuring thefluorescence of the fluo-Aβ42 (both freshly made and aggregated form) orfluorescein-labeled Escherichia coli K-12 BioParticles (fluo-E. coli)(Molecular Probe, Eugene, Oreg.) that are internalized. Cells arecultured in 24 well plates. To determine general phagocytic activityusing various concentrations of the bispecific antibodies (10 ng/ml, 25ng/ml, 50 ng/ml, 100 ng/ml 200 ng/ml, 400 ng/ml and 800 ng/ml) againstmonomer or oligomer and fibril Aβ treatments, cells are incubated withfluo-E. coli for 2 hours at room temperature. Trypan Blue is addedimmediately after removing the fluo-E. coli from cells to quench theextracellular probe. The wells of the plate are read by the WallacVictor2 1420 multilabel counter (Wallac, Md.) using 480 nm excitation,520 nm emission. Data is analyzed using Wallac Explore. For measuringthe effect of aggregation of Aβ, the fluo-Aβ is dissolved in DMSO thendiluted in serum free culture medium, and allowed to aggregate for 1hour at room temperature. The aggregated Aβ is vortexed, and sonicatedbefore being added to microglial cultures. The conditioned medium andcell lysate are measured by the Wallac Victor2 1420 multilabel counter.

c) Immunocytochemistry.

Immunocytochemistry of microglial cells is performed with polyclonalantibody Fc receptor gamma, and polyclonal MHCII. Cells are fixed with4% paraformaldehyde for 15 min. at room temperature, washed with PBSthree times, and then blocked with blocking buffer (2% BSA, 2% goatserum and 0.05% Triton X-100 in PBS) for 30 min. at room temperature.Cells are incubated with primary antibody for three days at 4° C. Thencells are rinsed with PBS three times and incubated with secondaryantibodies (Santa Cruz, Calif.) for 1 hour at room temperature. Toverify the internalization of Aβ by microglia, two sets of cells areincubated with unlabeled Aβ42 (Bachem, Calif.). Before blotting thecells with the anti-Aβ antibody (1:500 dilution, N-terminal 16-24)(Senetek, N.Y.), one set of cells is permeabilized with 0.1% TritonX-100 and the other without. A fluorescence microscope (Leica, Germany)with 25× and 100× objective will be used to observe a large number ofcells per field.

In Vivo Assay Using APP23 Transgenic Mice:

In Vivo Assays: Tests are performed to determine whether diabodiesdirected at clearing specific morphologies of Aβ can reduceamyloidogenic APP processing in transgenic mouse models of AD. As an invivo validation of the efficacy of the diabody constructs in clearing Aβand preventing plaque deposition, the diabodies are administeredintraperitoneally in transgenic mouse models of AD. The in vivo efficacyof administering the Aβ clearing diabodies as a means to reduce Aβtoxicity using similar methodologies is further tested. Five PSAPP/groupmice are chosen in gender matched groups for each treatment arm.Beginning at 8 weeks of age, PSAPP mice are i.p. injected with 100 μg ofthe appropriate diabody once every ten days for periods of 1, 2, and 3months. Initial test doses of these diabodies (1 μg to 1 mg) are triedin PSAPP mice to ensure that antibody penetration of the BBB occurs.Mouse brain is stained using a labeled anti-myc antibody such as 9E10 todetect penetration of i.p. injected diabody. The mice are sacrificed at3 months of age for analysis of pathology. A control diabody with noactivity to Aβ or clearance mechanisms is also be administered. Theeffect of the diabodies on APP processing is assessed by monitoring theextent of plaque deposition in the brain and the levels of the variousAPP proteolytic products, including Aβ, C99, and C83.

EXAMPLE 2

Aβ is proving to be a critical component in the etiology of AD.Accumulation of soluble Aβ has been correlated with the severity of ADand is thought to lead to diffuse plaque formation, setting off acascade of events including activation of microglial cells andinflammation. The combination of these events is thought to lead toneural dysfunction, cell death and misprocessing of tau, resulting inthe other dominant pathological feature of AD, neurofibrillary tangles.Furthermore, early-onset familial forms of AD are linked to mutations inthe amyloid precursor protein and presenilin genes, which play centralroles in the production and deposition of Aβ. Intermediate Aβmorphologies including small soluble oligomers and protofibrils havebeen implicated in AD. Soluble oligomers of Aβ were shown to becytotoxic, disrupt neuronal functions including LTP and learning, andcorrelate better with progression of AD and have been identified in ADbrains. Therefore well defined, highly specific reagents that canidentify individual morphologies of Aβ are a valuable tool for studyingand potentially treating AD. The inventors have developed a noveltechnology that utilizes AFM to identify specific protein morphologiesand simultaneously isolate scFvs that bind the target morphology. Inorder to apply this technology to isolating scFvs against specific Aβmorphologies we first need to refine the panning protocols to favorselection for each of the various Aβ forms.

Inhibition of Aβ Aggregation with scFvs.

The inventors isolated scFvs to different regions of full length Aβ anddemonstrated that the scFvs can successfully inhibit aggregation andtoxicity of AP. The inventors characterized two scFvs against Aβ anddefined their binding epitopes by immobilizing various Aβ peptides to abiosensor surface and testing for binding. It has been demonstrated thatthe scFvs could inhibit Aβ aggregation utilizing both Thioflavinfluorescence assays and AFM image analysis. It was showed that we couldimage various Aβ morphologies by AFM, and that we could quantify thedistribution of oligomeric aggregate sizes. It was also demonstratedthat toxic effects of Aβ aggregation on the neuroblastoma cell line,SH-SY5Y could be alleviated by incubation with scFvs.

It was further identified that critical regions of Aβ that control theaggregation state of the protein. We incubated various peptide fragmentsof Aβ both alone and in conjunction with Aβ and showed that residues17-20 and 30-35 play essential roles in the aggregation process. Inaddition, we showed that the 25-35 peptide, previously shown to behighly toxic to cells, promotes formation of oligomers and protofibrilsin Aβ42, but not fibrils, and the peptide by itself forms smalloligomeric structures and very thin, long filaments. These resultsprompted the inventors to isolate scFvs to the 25-35 region of Aβ totest how they alter aggregation and toxicity. It was shown that scFvsgenerated against this region are very effective at reducing bothaggregation and cytotoxicity (Zameer et al. 2006).

Affinity Maturation of Antibodies to Aβ.

The inventors identified a scFv with the modest affinity for Aβ (highnanomolar affinity), H1 (see FIG. 7), and used this scFv as a parentsequence for affinity maturation studies to generate scFvs withincreased affinity for Aβ. The inventors have successfully increased theaffinity of the parent H1 scFv approximately 5-fold (K_(D) ofH1v2=2.47×10-7 M) through standard affinity maturation protocols (Schieret al. 1996). The inventors have developed a modified protocol using aBIACore biosensor to directly select for scFvs with slower off-rates,and have isolated scFvs with 10-fold better K_(D) values than the H1v2clone isolated from the same library using the standard protocol (Yuanet al. 2006). The inventors have also utilized the yeast display libraryto isolate scFvs against biotinylated peptides at their N-terminal ofα-synuclein, experiments that are essentially identical to those thatcan be performed for affinity maturation studies. After four rounds ofmagnetic bead enrichment and FACS sorting as described in the methodssection, over 10% of the cells show positive binding as shown with oneof the target antigens (FIG. 4).

Isolating Morphology Specific ScFvs.

An increasing number of studies suggest that a number of differentneurological diseases, i.e. AD, PD, Huntington and ALS, share a commonpathological mechanism, namely, a misfolded protein that aggregates andleads to a dysfunction in the central nervous system (Forman et al.2004)). The proteins accumulate as fibrillar β-pleated sheet structuresin specific regions and cells of the brain depending on the protein anddisease involved. All the proteins appear to follow similar pathways inthe aggregation process, first increasing β-sheet content, then formingsmall oligomers and protofibrils intermediates, and finally formingfibrillar amyloid deposits. In order to define the role of theseintermediates in the various diseases, it would be extremely beneficialto have highly specific reagents to identify the different proteinforms. Toward this goal, the inventors have developed a biopanningtechnology to enable the isolation of scFvs that bind specificmorphologies. The inventors have combined the imaging capabilities ofAFM with phage display antibody technology to allow the identificationof the presence of a specific protein morphology and then isolate scFvsthat bind that morphology (Barkhordarian et al. 2006). The basicprotocol is illustrated in FIG. 5.

To demonstrate the capabilities of this technology, scFvs were isolatedthat bind either oligomeric or fibrillar morphologies of α-synuclein andAβ. First, incubation conditions were identified that favor formation ofeither oligomers or fibrils. To verify the protein morphology, analiquot (10 μl) of the incubated solution was deposited on freshlycleaved mica and fixed for 5 minutes. Then the substrate was washedthree times with 1 ml of ultra-pure water. The sample was then driedunder a gentle stream of argon gas and imaged by AFM. Using thisprotocol the inventors were able to reliably reproduce oligomeric andfibrillar samples (see FIGS. 6A and 6B).

Utilizing the novel AFM biopanning technology (see FIG. 5), theinventors isolated scFvs against fibrillar (Barkhordarian et al. 2006)and oligomeric a-syn (Emadi et al. 2007) forms. For both panning cases,after only two rounds of biopanning, around 50% of recovered clonesindicated positive binding to the desired target in a preliminary ELISAtest. One of the stronger binding clones was selected based on thepreliminary test and showed that the anti-oligomeric a-syn scFvspecifically bound only an oligomeric form of a-syn, and not monomericor fibrillar forms. We also showed that the anti-oligomer scFv couldinhibit formation of fibrils, but not oligomers, that oligomeric, butnot monomeric or fibrillar a-syn when added extracellularly to aneuronal cell line exhibited cytotoxicity, and that the anti-oligomericscFv could inhibit toxicity of preformed a-syn oligomers (Emadi et al.2007). We have also isolated a second scFv against a larger oligomericform of a-syn. The two different anti-oligomeric scFvs do not show crossreactivity, the scFv binding the smaller earlier stage oligomers doesnot react with larger later stage oligomers, and the scFv against largerlater stage a-syn oligomers does not react with the smaller, earlierstage oligomers (FIGS. 6A and 6B). In addition the anti-syn oligomericscFvs do not cross react with Aβ oligomers. Significantly, both theanti-syn oligomer scFvs recognized aggregates in human tissue samplestaken from Parkinson's brains, but not from Alzheimer's or controlbrain.

The inventors have also isolated an scFv against oligomeric Aβ. The scFvrecognizes a larger later stage oligomeric form of Aβ. The scFvs againstoligomeric Aβ did not cross react with a-syn oligomers and also labeledsamples taken from human AD brain tissue, but not Parkinson's brain orcontrol brain tissue. The anti-oligomeric Aβ scFv also inhibitedaggregation and toxicity of Aβ towards SH-SY5Y cells (Zameer et al.2008).

Aβ Aggregation.

Soluble oligomers of Aβ are formed as transient intermediates during theaggregation of monomeric Aβ to fibrillar amyloid. In order toreproducibly form Aβ aggregate morphologies, it is critical to removeany preformed Aβ aggregate seeds. The inventors dissolve lyophilized Aβin hexafluoroisopropanol (HFIP), centrifuge, aliquot into Eppendorftubes and then dry with nitrogen as described (Liu et al. 2004; Liu etal. 2005) to prepare standard monomeric Aβ stock solutions. Aβ oligomerscan be formed by dissolving the Aβ stock solutions in solution with DMSOand diluting in buffer as described (Liu et al. 2004; Liu et al. 2004)or dissolving in HFIP and diluting (Kayed et al. 2003; Stine et al.2003). Increasing concentrations of Aβ form fibrillar aggregates morequickly, while increasing concentrations of DMSO will decrease theaggregation rate.

AFM Imaging.

AFM images surfaces by scanning them in a raster pattern with a finetip. The AFM tip follows the contour of surface structure which is thendepicted on a computer screen directly during scanning. The inventorshave found that deposition of the aggregate samples on unmodifiedfreshly cleaved mica allows the ability to obtain high quality images ofthe protein aggregates. The main advantage of using bare mica however isthat phage particles do not bind to this surface well, and unbound phagecan be readily washed off as described in the preliminary resultssection.

Phage Preparation.

The Tomlinson I and J scFv libraries supplied by the Cambridge AntibodyTechnology (Cambridge, England) are used for phage preparation. Bothlibraries (I and J) are based on a single human framework for VH and VLand use pIT2 as a phagemid vector. The CDR3 of the heavy chain wasdesigned to be as short as possible yet still allow for antigen binding.Side-chain diversity was incorporated in CDR3 and CDR2 regions atpositions which make contacts to the antigens and are highly diverse intheir mature native repertoire. CDR1 regions are kept constant. The sizeof each library is about 1.4×10⁸. The libraries contain clones that werepre-selected for active folding domains by binding to Protein-L andProtein-A. Both libraries are grown separately, and their phage mixed1:1 for selection protocols. Phage is produced from the initialbacterial library stock by superinfection of the bacterial culture withhelper phage KM13 as described by CAT. Phage samples are purified fromthe supernatant by polyethylene glycol (PEG) precipitation andresuspended in PBS (phosphate-buffered saline) and used for panning.

AFM Panning and Selection.

The AFM biopanning and selection protocols to isolate scFvs againstspecific morphologies of a-syn and are performed essentially asdescribed (Barkhordarian et al. 2006; Emadi et al. 2007; Zameer et al.2008).

Binding Kinetics.

Antibody binding studies are performed by surface plasmon resonanceusing a BIAcore X biosensor. Surface plasmon resonance is based on theprinciple that light waves at a metal surface can be resonantly coupledinto electric oscillations, or surface plasmons. The surface plasmonsgenerate an evanescent wave that decays with increasing distance fromthe metal surface. Protein interactions at the surface alter theevanescent wave and plasmon characteristics, changing the internallyreflected light signal. This change in reflected light can bequantitatively monitored using a diode array. To test for bindingaffinities to the various Aβ morphologies, the different scFvs areindividually immobilized on carboxymethyl dextran sensor chips (CM5,Biosensor) by amine coupling. The dextran surface of the sensor chip isfirst activated to produce N-hydroxysuccinimide esters usingN-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-diethylaminopropyl)Carbodiimide (EDC) (O'Shannessy et al. 1992). The scFv is thenimmobilized to the activated surface through primary amine groups. Asolution containing one of the different Aβ morphologies is injectedover the peptide surface at a rate of 5 μL/min. Bound Aβ is then allowedto dissociate completely by flowing buffer (PBS with 0.005% Tween) overthe surface before the next sample is injected. The two rate constants,k_(on) and k_(off), and the corresponding equilibrium dissociationconstant, K_(d)=k_(off)/k_(on), can be obtained by curve fitting of theobtained data. In this manner the affinity of each scFv for eachmorphology of Aβ or for any other protein aggregate morphology isaccurately calculated. If the calculated dissociation constant for aparticular antibody is not sufficiently high enough for in vivo studies,the affinity of the antibody is increased by generating a secondaryantibody library and rescreened.

Aggregation Kinetics.

Aβ aggregation in the presence of the different scFvs is monitored byThioflavin fluorescence and AFM analysis. Thioflavin fluorescence isuseful for monitoring fibril formation while AFM studies can determinethe extent of fibrillar and oligomeric aggregation. AFM analysis isperformed to determine the size distribution of the aggregated particlesas well as to characterize antibody specificity.

Cytoxicity Assay.

Cell toxicity of the various aggregate samples with and without addedscFv is tested using two different toxicity assays, reduction inmitochondrial activity using an MTT assay, and increase in releasedlactate dehyrogenase activity using an LDH assay. These assays have beenpreviously described (Emadi et al. 2004; Liu et al. 2004; Liu et al.2004; Liu et al. 2005; Zameer et al. 2006; Emadi et al. 2007; Nannengaet al. 2008; Zameer et al. 2008).

Affinity Maturation.

Phage Libraries: Antibodies with picomolar affinities for the differentAβ morphologies are sufficiently specific for use either as a diagnosticfor detecting the presence of the individual morphologies in AD samplesor as a potential therapeutic for targeting specific Aβ morphologies invivo. Aβ is normally present in CSF at low nanomolar concentrations, soan antibody with picomolar affinity should readily bind a sufficientamount of soluble Aβ at normal levels. While it is not known what theconcentration of oligomeric forms of Aβ are in vivo, the scFvs can beaffinity matured to higher affinity if necessary. Antibodies withnanomolar affinities toward fibrillar Aβ were shown to successfullyinitiate antibody mediated clearance of Aβ deposits (Bard et al. 2000).To generate antibodies with picomolar affinity, in certain embodimentsaffinity maturation techniques may be utilized. A CDR region of each ofthe antibody light chain and heavy chain fragments is separatelyrandomized and the chains are randomly shuffled and recombined togenerate a secondary library of antibodies where the entire library isnow based on antibody sequences specific for Aβ. The first generation ofthis library is constructed by randomizing the CDR3 region of the lightchain essentially as described (Schier et al. 1996). The best bindersfrom this new library is selected by panning as described above. ThosescFvs with the highest affinity serve as the framework for the secondgeneration library formed by randomizing the CDR2 regions of the heavyand light chain genes again essentially as described (Schier et al.1996) and shuffling the light and heavy chains. The best binders fromthis second generation library are isolated and serve as the frameworkfor the third generation library formed by randomizing the CDR1 regionsof the light and heavy chains. With each generation scFvs are isolatedwith better and better affinity for Aβ. This type of affinity maturationis continued until scFvs with picomolar affinity for the differentmorphologies of Aβ are isolated. Affinity maturation of phage displaypeptides has routinely improved the affinity of the parent antibody(Jackson et al. 1995; Irving et al. 1996; Schier et al. 1996; Wu et al.1998; Chowdhury and Pastan 1999; Olsen et al. 2000), even into thefemtomolar range (Boder et al. 2000).

In addition, in certain embodiments, affinity mature scFvs are developedso that they bind not just a specific morphology, but a specificmorphology of a specific protein. Current oligomeric antibodiesrecognize oligomeric structures of many different proteins (Kayed et al.2003). While there are some advantages to such an antibody, there arealso disadvantages in that these antibodies cannot distinguish thepresence of insulin, Aβ, or synulein oligomers. With the present AFMpanning protocol it is possible to select scFvs for a specific proteinmorphology form by using a two-step negative/positive selection. Forexample, one can generate a secondary library of scFvs againstoligomeric Aβ. Using this secondary library, one can then pan for scFvsthat are specific only for Aβ oligomers by modifying the panningprotocol. One first performs a negative panning step by incubating thescFv library with the non-desired oligomeric proteins, (for example;synuclein, huntingtin, prion) to remove scFvs that recognize theseaggregates. One then perform the positive panning step by taking theremaining non-bound phage and select for those that bind Aβ oligomers.The inventors have successfully utilized this two-step panning protocolto isolate scFvs to a specific protein among a family of relatedproteins (Wu et al. 1998).

Affinity maturation can be performed using phage display libraries oryeast display libraries. Each of these libraries has particularadvantages for different situations. For oligomeric morphologies of Aβwhere AFM must be used for the selection protocol, phage displaylibraries have to be used for the panning process. For monomeric orfibrillar morphologies where the protein can be tagged with biotin,yeast display libraries can be used. The advantage of yeast displaylibraries is that high affinity variants can be directly selected duringthe flow cytometry sorting process. Therefore both of these affinitymaturation protocols are described below.

Affinity maturation of phage display antibodies has routinely improvedthe affinity of the parent, even evolving to obtain femtomolar affinity.The protocols for generating these second generation antibody librariesare described in the various references (Jackson et al. 1995; Irving etal. 1996; Schier et al. 1996; Wu et al. 1998; Chowdhury and Pastan 1999;Olsen et al. 2000). Basically, a second generation library will beconstructed essentially as described (Kim and Tanzi 1997) with someminor modifications as follows. The CDR3 light chain region of theparent H1 scFv is randomized using a two-step PCR protocol. The scFvgene is amplified in the first PCR using the two primers: LMB3 (5′-CAGGAA ACA GCT ATG AC-3′ (SEQ ID NO: 3)) and CDR3-6-VL-FOR (5′-CTT GGT CCCTCC GCC GAA TAC CAC NNN NNN NNN NNN NNN NNN AGA GGA GTT ACA GTA ATA GTCAGC CTC-3′ (SEQ ID NO: 4)) where N represents a random nucleotide. Theresulting PCR product has an Nco I restriction endonuclease site at the5′ end. In the second PCR step, the randomized CDR3 light chainfragments are further amplified using the following two primers: LMB3(5′-CAG GAA ACA GCT ATG AC-3′ (SEQ ID NO: 3)) and JL-NOT-FOR (5′-ATT GCTTTT CCT TTT TGC GGC CGC GCC TAG GAC GGT CAG CTT GGT CCC TCC GCC-3′ (SEQID NO: 5)) to introduce a Not I restriction endonuclease site at the 3′end. The PCR product obtained after the two PCR steps is digested withboth Nco I and Not I, and ligated into plasmid pHEN2, which ispreviously digested with Not I and Nco I. The ligated mixture istransformed into electrocompetent E. coli TG1 cells by electroporationto yield the second-generation phage display library. The secondgeneration library is then utilized in biopanning experiments asdescribed above. Additional library generation can be constructed byvarying different CDR regions. While the primers will vary to flank thetarget CDR sequence, the protocols are essentially the same.

Affinity Maturation with Yeast Libraries.

Yeast surface display of scFv antibodies can also be used to isolatehigher affinity clones from small (˜1×10⁶) mutagenic libraries generatedfrom a unique antigen binding scFv clone (Boder et al. 2000). Thesecondary mutagenic libraries are constructed by amplifying the parentalscFv gene using error-prone PCR to incorporate 3 to 7 pointmutations/scFv (Stemmer 1994; Daugherty et al. 2000). The material iscloned into the surface expression vector using the endogenoushomologous recombination system present in yeast, known as “Gap-Repair”(On-Weaver and Szostak 1983). Gap repair is an endogenous homologousrecombination system in S. cerevisiae that allows gene insertion inchromosomes or plasmids at exact sites by utilizing as little as 30 basepair regions of homology between the gene of interest and its targetsite. This allows mutated libraries of 1-10×10⁶ clones to be rapidlygenerated and screened by selecting the brightest antigen bindingfraction of the sorted cell population using decreasing amounts ofantigen relative to the KD of the starting parental clone. The screeninginvolves 3 to 4 rounds of flow cytometry sorting as described above.Flow cytometry will either be performed at the shared user facility tobe housed in the BIO-Design facility at ASU or through the continuingcollaborative arrangement with Barrow's Neurological Institute where allof the preliminary data was obtained.

Although the foregoing specification and examples fully disclose andenable the present invention, they are not intended to limit the scopeof the invention, which is defined by the claims appended hereto.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms (Le., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

CITATIONS

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What is claimed is:
 1. A bispecific antibody fragment comprising (a) afirst scFv that is specific for soluble Aβ, wherein the first scFv isH1v2 anti-Ab40 (SEQ ID NO: 6), H1v3 anti-Ab40 (also called D9antiAb40)(SEQ ID NO: 7), or C1 antiAb40 (SEQ ID NO: 8), and (b) a second scFvthat specifically activates a classical complement pathway component ordirectly activates microglial cells, wherein the scFv fragmentspecifically binds to C1q.
 2. An antibody construct comprising thebispecific antibody fragment of claim 1, further comprising a secondbispecific antibody fragment.
 3. The bispecific antibody fragment ofclaim 1, further comprising a (Gly₄-Ser)₃ linker (SEQ ID NO: 1) operablylinked between the first and second scFvs.
 4. The bispecific antibodyfragment of claim 3, further comprising a poly-His tail operably linkedto either the first or second scFvs.
 5. The bispecific antibody of claim1, wherein (a) the first scFv is an H1 scFv specific for β-amyloid,wherein the first scFv has an amino-terminus and a carboxy-terminus, and(b) the second scFv is an scFv specific for C1q, wherein the second scFvhas an amino-terminus and a carboxy-terminus, and wherein the bispecificantibody further comprises (c) a (Gly₄Ser)₃ peptide linker (SEQ IDNO: 1) operably linking the carboxy-terminus of the first scFv to theamino-terminus of the second scFv.
 6. The bispecific antibody of claim1, further comprising a gIII signal sequence operably linked to theamino-terminus of the first scFv.
 7. The bispecific antibody of claim 1,further comprising a c-myc tag operably linked to the carboxy-terminusof the second scFv.
 8. The bispecific antibody of claim 7, furthercomprising a (His)₆ tag (SEQ ID NO: 2) operably linked to the c-myc tag.9. A nucleic acid encoding the bispecific antibody of claim
 1. 10. Anexpression cassette comprising the nucleic acid sequence of claim 9, anda promoter.
 11. A vector comprising the expression cassette of claim 10.12. A cell comprising the nucleic acid of claim
 9. 13. A therapeuticcomposition comprising the bispecific antibody of claim 1, incombination with a physiologically-acceptable, non-toxic vehicle.
 14. Amethod of clearing aggregated and soluble Aβ comprising administeringthe bispecific antibody of claim
 1. 15. The method of claim 14, whereinthe aggregated and soluble Aβ is in a cell.
 16. The method of claim 15wherein aggregated and soluble Aβ is in brain tissue.
 17. The method ofclaim 16, wherein the brain tissue is in a mammal.